The Crc Global Regulator Inhibits the Pseudomonas putida pWW0 Toluene/Xylene Assimilation Pathway by Repressing the Translation of Regulatory and Structural Genes

Abstract

In Pseudomonas putida, the expression of the pWW0 plasmid genes for the toluene/xylene assimilation pathway (the TOL pathway) is subject to complex regulation in response to environmental and physiological signals. This includes strong inhibition via catabolite repression, elicited by the carbon sources that the cells prefer to hydrocarbons. The Crc protein, a global regulator that controls carbon flow in pseudomonads, has an important role in this inhibition. Crc is a translational repressor that regulates the TOL genes, but how it does this has remained unknown. This study reports that Crc binds to sites located at the translation initiation regions of the mRNAs coding for XylR and XylS, two specific transcription activators of the TOL genes. Unexpectedly, eight additional Crc binding sites were found overlapping the translation initiation sites of genes coding for several enzymes of the pathway, all encoded within two polycistronic mRNAs. Evidence is provided supporting the idea that these sites are functional. This implies that Crc can differentially modulate the expression of particular genes within polycistronic mRNAs. It is proposed that Crc controls TOL genes in two ways. First, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes. Second, Crc inhibits the translation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation. This ensures a rapid inhibitory response that reduces the expression of the toluene/ xylene degradation proteins when preferred carbon sources become available.

Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa

“…CrcZ has high affinity for the Crc protein and, when in excess, prevents Crc from acting as a translational repressor of target mRNAs. Typical Crc targets are mRNAs encoding porins, uptake systems, and enzymes involved in the degradation of less-preferred substrates (20,27,28). In P. aeruginosa, CrcZ levels are lower during growth on succinate than during growth on less-preferred substrates such as glucose or mannitol, reflecting the activity of the CbrA/CbrB system as a master regulator of carbon catabolite repression (36).…”

Catabolite Repression Control of Pyocyanin Biosynthesis at an Intersection of Primary and Secondary Metabolism in Pseudomonas aeruginosa

“…The Crc protein (subunit size, 28.5 kDa) is a global regulator whose structure has not yet been elucidated. Crc recognizes an A-rich ribonucleotide sequence, which we have termed the CA motif (consensus AAnA AnAA, where n is preferentially C or U) and which is typically located near the ribosome binding sites of target mRNAs (17,18,19,26). In Pseudomonas putida, but not in P. aeruginosa, CrcZ is assisted by CrcY, a second Crc-binding sRNA (20).…”

Regulation of Bacterial Metabolism by Small RNAs Using Diverse Mechanisms

“…Crc binding has no apparent effect on mRNA stability but prevents formation of translation initiation complexes. First in P. aeruginosa (118) and subsequently in other pseudomonads (73), an ∼400-nt noncoding RNA, CrcZ, was identified as a crucial regulator of Crc activity. CrcZ binds Crc with high affinity and sequesters it, preventing its interaction with mRNAs.…”